CN111789028A

CN111789028A - Sporotrichum ciresi propagation method and application thereof

Info

Publication number: CN111789028A
Application number: CN202010793692.5A
Authority: CN
Inventors: 李良波; 黄荣韶; 姜学松; 宁珊; 吴飘
Original assignee: Jiangxi Hongxing Pharmaceutical Co ltd
Current assignee: Jiangxi Hongxing Pharmaceutical Co ltd
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2020-10-20

Abstract

The invention relates to the technical field of Postophylla pyrrosia propagation, and particularly discloses a sporophyte propagation method of Postophylla pyrrosia and application thereof. By adopting the method, a large amount of prothallium materials can be obtained, two stages of subculture and rooting culture of the sporophytes are not needed in the culture process, the induction period of the sporophytes is shortened, and only 60 days are needed from spore germination to juvenile sporophyte formation; in addition, the induction rate of sporophyte is improved, pyrrosia peduncularis plants can be quickly obtained, theoretical and technical support is provided for industrial seedling raising, the problem that the conventional pyrrosia peduncularis propagation method is mostly based on plant division propagation and has a long culture period is solved, and the method has a wide market prospect.

Description

Sporotrichum ciresi propagation method and application thereof

Technical Field

The invention relates to the technical field of pyrrosia peduncularis propagation, in particular to a sporophyte propagation method of pyrrosia peduncularis and application thereof.

Background

Pyrrosia petiolata (Christ) Ching is a saprophytic (Polypodiaceae) Pyrrosia genus (Pyrrosia) type pteridophytes, the back of sporophyll is covered by thick layer of hairy star, and a large number of sporangium groups are arranged. The pyrrosia petiolata has the effects of inducing diuresis for treating stranguria, clearing away damp-heat and the like, and has higher medicinal value, so the demand of pyrrosia petiolata is continuously increased. However, in recent years, wild resources are reduced along with the increasing clinical dosage of pyrrosia petiolata, so that artificial cultivation is required to improve the breeding efficiency. The research of factors influencing the conversion of GGB (green globular pellets) into sporophytes from the aspect of culture conditions is the key of rapid propagation of pyrrosia pedunculata plants.

At present, researches on propagation methods of pyrrosia peduncularis are mostly carried out by means of plant division propagation, the culture period is long, generally, the culture period is more than two months, and no related report about culture of pyrrosia peduncularis sporophytes is found. The tissue culture has the characteristics of high propagation speed, high propagation coefficient and the like, but only a large number of prothallium bodies can be obtained in the spore tissue culture research of many pyrrosia plants, and juvenile sporophytes are difficult to obtain. Therefore, the establishment of the pyrrosia petiolata sporophyte propagation system can provide theoretical and technical support for the industrial seedling culture.

Disclosure of Invention

The embodiment of the invention aims to provide a propagation method of pyrrosia peduncularis sporophytes, which aims to solve the problems that the existing propagation method of pyrrosia peduncularis in the background art is mostly based on plant division propagation and has a long culture period.

In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

a sporophyte propagation method of pyrrosia petiolata comprises the following steps:

1) selecting mature pyrrosia pedunculata sporophyll, and obtaining spore powder from the sporophyll after disinfection to obtain a disinfected explant material;

2) inoculating the explant material obtained in the step 1) into a germination culture medium for pyrrosia pedunculata spore germination to obtain a flaky prothallium; wherein the germination culture medium is a culture medium obtained by diluting an MS culture medium (Murashige and Skoog culture medium) without adding cane sugar into different concentrations, and the pH value of the germination culture medium is 5.6-6.0;

3) transferring the sheet-shaped prothallium obtained in the step 2) to a proliferation culture medium for proliferation to obtain a prothallium group; the proliferation culture medium is designed to be different in concentration of sucrose solution and B₅Culture medium 1/2B₅The culture medium is combined to obtain the culture medium B₅Culture medium B designed in 1968 and used in Gamborg et al (Gamborg)₅A culture medium, wherein the pH value of the proliferation culture medium is 5.6-6.0;

4) and (3) carrying out illumination induction on the prothallium group obtained in the step 3) to obtain a juvenile sporophyte group.

The pyrrosia pedunculata sporophyll is mature sporophyll with plump and robust sporangia collected in Anshu city of Guizhou in 2018 for 10 months, impurities on the surface of the sporophyll are slightly swept down by a brush, and the sporophyll is filled into a sulfuric acid paper bag and stored in a refrigerator at 4 ℃ for later use. The whole disinfection process is carried out on a super-clean workbench, and the filter paper and the inoculation tool are sterile.

As a further scheme of the invention: the sterilization is to use alcohol, sterile water and HgCl to sterilize mature pyrrosia pedunculosa sporophyll₂The solution is sterilized.

Preferably, the sterilization is carried out by using mature pyrrosia petiolata sporophyll with volume concentration of 70%Soaking in ethanol for 30s, washing in sterile water for 3 times, and adding 0.1 wt% of HgCl₂Soaking in the solution for 8min, and washing with sterile water for 5 times.

As a still further scheme of the invention: the spore powder is obtained by firstly absorbing water on the surface of the spore leaf by using sterile filter paper and then gently scraping the spore powder from the spore leaf by using a sterile scalpel to obtain a sterilized explant material.

As a still further scheme of the invention: the mature pyrrosia petiolata sporophyll is selected, impurities on the surface of the pyrrosia petiolata sporophyll are slightly swept down by a brush, and the pyrrosia petiolata sporophyll is put into a sulfuric acid paper bag and stored in a refrigerator at 0-10 ℃ (preferably 4 ℃) for later use.

Preferably, the pH of the germination medium is 5.8.

Further preferably, the germination medium is prepared by adding agar at 6g/L to 1/8MS medium.

As a still further scheme of the invention: the germination is carried out by adopting a daylight lamp light source at the culture temperature of 25 +/-2 ℃, the illumination intensity is 1000-.

Preferably, the culture temperature of the germination is 25 +/-2 ℃, a daylight lamp light source is adopted for illumination, the illumination intensity is 1000-.

As a still further scheme of the invention: the sucrose solution with different concentrations has a concentration of 0-30 g.L^-1The sucrose solution of (1).

As a preferable scheme: the proliferation medium is B₅The culture medium prepared by the culture medium, 30g/L of sucrose and 6g/L of agar is the optimal proliferation culture medium for the sheet-shaped prothallium.

As a still further scheme of the invention: the proliferation is carried out by adopting a daylight lamp light source at the culture temperature of 25 +/-2 ℃, the illumination intensity is 1000-.

Preferably, the culture temperature for proliferation is 25 +/-2 ℃, a daylight lamp light source is adopted for illumination, the illumination intensity is 1000-.

As a still further scheme of the invention: the illumination induction is specifically to perform illumination by adopting a daylight lamp light source at the culture temperature of 25 +/-2 ℃, the illumination time is 10-14h/d, the illumination intensity is 1000-.

Preferably, the light induction is to irradiate with a daylight lamp light source at a culture temperature of 25 +/-2 ℃, the light irradiation time is 12h/d, the light irradiation intensity is 1000-.

As a still further scheme of the invention: before the irradiation induction of the prothallium mass, the method also comprises the step of hardening seedlings of the prothallium mass.

As a still further scheme of the invention: the prothallium mass further comprises, before illumination induction: and (3) hardening the seedlings of the prophyll clusters for 3-5 days, taking out GGB (green cluster-shaped pellets) from the proliferation culture medium, cleaning the culture medium at the base part GGB, and planting the cleaned culture medium in a plug tray filled with vermiculite.

Preferably, the vermiculite is planted in a plug tray filled with the vermiculite, and is subjected to sterilization treatment, namely high-pressure steam sterilization at 121 ℃ for 30min, and the vermiculite is placed in an oven to be dried after the sterilization is finished; the surface of the plug tray is disinfected by 5% sodium hypochlorite solution before use, the sterilized vermiculite is filled after being dried, the plug tray is compacted and leveled, and the whole basin is soaked until the surface of the substrate is wet.

As a still further scheme of the invention: the method for propagating the sporophyte of pyrrosia peduncularis also comprises the step of transplanting, wherein the transplanting is to separate juvenile sporophyte groups obtained by illumination induction into single juvenile sporophytes, transplant the single juvenile sporophytes into a matrix, and carry out watering conventional management to obtain pyrrosia peduncularis plants.

As a still further scheme of the invention: the substrate is one or more of river sand, garden soil, quartz sand, vermiculite, pine needle soil, bark or perlite.

Preferably, the matrix is one of the following 6 matrices: A. river sand; B. soil preparation; C. quartz sand; D. vermiculite; E. river sand: garden soil: pine needle soil is 1:1: 3; F. vermiculite: bark: perlite is 2:1: 1.

As a still further scheme of the invention: the transplanting into the matrix is to transplant the single juvenile sporophyte into the matrix according to the row spacing of 2-3 cm.

Preferably, the transplanting into the matrix is transplanting the single juvenile sporophytes into the matrix at a row pitch of 2.5 cm.

As a still further scheme of the invention: after transplanting into the matrix, watering the matrix thoroughly, covering the pot mouth with a plastic film, uncovering the film after the young plants are adapted, and carrying out conventional management.

Another object of the embodiments of the present invention is to provide the application of said method for propagation of sporophytes of pyrrosia petiolata in cultivation of ferns.

As a still further scheme of the invention: in the application of the method for propagating the sporophyte of the pyrrosia peduncularis in the cultivation of the pteridophyte, when the pteridophyte is cultivated, the pyrrosia peduncularis can be directly replaced by the corresponding pteridophyte for cultivation, and of course, specific parameters can be flexibly adjusted according to needs, and the method is not limited herein; the ferns are generally divided into 5 subgenera: the specific species of the conidia ferdii (Psilophytina), the Sphenophytina (Sphenophytina), the Lycophytina, the allium tuberosum (Isoephytina) and the pteridophytina real (Filicophytin) are selected according to the requirements, and are not limited herein.

Compared with the prior art, the invention has the beneficial effects that:

the embodiment of the invention provides a method for propagating sporophyte of pyrrosia petiolata, which comprises the steps of inoculating sterile pyrrosia petiolata spores into a germination culture medium without adding cane sugar for germination, transferring the culture medium to a proliferation culture medium for proliferation, and then obtaining pyrrosia petiolata sporophyte through seedling exercising treatment and illumination induction. By adopting the method, a large amount of prothallium materials can be obtained, and the spore germination rate is 100%; two stages of subculture and rooting culture of sporophytes are not needed in the culture process, the induction period of the sporophytes is shortened, and only 60 days are needed from spore germination to juvenile sporophyte formation; moreover, the induction rate of the sporophyte is improved and reaches up to 85 percent; the pyrrosia peduncularis plant can be quickly obtained, the transplanting survival rate of the juvenile sporophytes is 100%, theoretical and technical support is provided for industrial seedling raising, the problem that the conventional pyrrosia peduncularis propagation method is mostly based on plant division propagation and has a long culture period is solved, and the pyrrosia peduncularis propagation method has a wide market prospect.

Drawings

FIG. 1 is a schematic diagram of Podostigma longituba spore germination in example 1.

FIG. 2 is a schematic diagram showing the proliferation of Podostigma longituba prothallium in example 1.

FIG. 3 is a schematic diagram showing the induction of the sporophyte of pyrrosia petiolata in example 1.

FIG. 4 is a schematic diagram showing the transplantation of the sporophytes of pyrrosia petiolata in example 1.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1

(1) selection and disinfection of pyrrosia pedunculata spores

Collecting mature sporophylls with full and robust sporangiums in Anshun city of Guizhou in 2018 for 10 months, slightly sweeping impurities on the surfaces of the sporophylls off by using a brush, collecting the sporophylls, filling the sporophylls into a sulfuric acid paper bag, and storing the sporophylls in a refrigerator at 4 ℃ for later use; sterilizing sporophyll on a clean bench, soaking in 70% ethanol for 30s, washing in sterile water for 3 times, and adding 0.1 wt% HgCl₂Soaking in the solution for 8min, and washing with sterile water for 5 times; the surface water of the spore leaf is firstly absorbed by sterile filter paper, and then the spore powder is gently scraped off from the spore leaf by a sterile scalpel, so as to obtain the sterilized explant material.

(2) Pyrrosia peduncularis spore germination culture

Inoculating the sterilized explant material into a germination culture medium prepared by adding agar 6g/L (namely adding 6g of agar per liter of culture medium) into 1/8MS culture medium for pyrrosia petiolata spore germination to obtain a flaky proleaf body, wherein the germination culture medium is not added with sucrose and has a pH value of 5.8. The culture temperature of germination is 25 ℃, the illumination intensity is 1000-. And inoculating 5 bottles of seeds for each germination culture, repeating for 3 times, and observing and recording the germination condition of the spores. Germination time refers to the time when small green spots appear on the medium, and specific germination results are shown in table 1.

TABLE 1 Germination results table

(3) Postophyllia tenosa prothallium proliferation

Transferring the obtained sheet-like prothallium to a proliferation medium (30 g. L) for proliferation to obtain a prothallium mass^-1Sucrose solution and B₅The culture medium combination, each liter of multiplication culture medium is added with 6g of agar, the pH value is 5.8, the multiplication culture temperature is 25 +/-2 ℃, the illumination intensity is 1000-. Specific proliferation results are shown in table 2. FIG. 2 is a photograph showing proliferation of Podostigma longipes in example 1.

TABLE 2 Protozoa proliferation results

(4) Podostigmus petiolatus induction

Placing vermiculite as culture medium in a high pressure steam sterilization pot at 121 deg.C for 30min, and drying in an oven after sterilization. Before the seedling raising tray is used, a 5% sodium hypochlorite solution is used for surface disinfection, the sterilized culture medium is filled in the seedling raising tray after being dried, the culture medium is placed in the seedling raising tray and then compacted and leveled, and the whole tray is soaked in water until the surface of the culture medium is wet. Taking out the proliferated prothallium mass from the culture bottle, washing the culture medium with distilled water, transplanting the prothallium mass into a matrix for illumination induction to obtain juvenile sporophyte mass, and setting the illumination period at 12h/d and the illumination intensity at 2000lx for treatment. Spraying water once a day. Periodically observing the illumination induction condition of the sporophyte, recording the initial time of forming the sporophyte and counting the induction rate. Sporulation time refers to the time from the mass of the original leaf until the appearance of the first true leaf. Specific sporozoite induction results are shown in table 3. FIG. 3 is a photograph showing the induction of the sporophyte of pyrrosia petiolata in example 1.

TABLE 3 sporozoite Induction results

(5) Podostigmus petiolatus sporophyte transplantation

Separating the juvenile sporophyte groups obtained by induction into single juvenile sporophytes, and transplanting the single juvenile sporophytes into a matrix, wherein the matrix is vermiculite: bark: perlite is 2:1: 1. Transplanting the juvenile sporophytes into the matrix at the row spacing of 2.5cm, watering the matrix thoroughly, covering the pot mouth with a plastic film, uncovering the film after the juvenile plants are adapted, and carrying out conventional management. After 30d, recording the survival rate of juvenile sporophytes. Specific sporozoite transplantation results are shown in table 4. FIG. 4 is a photograph showing the presence of transplantation of pyrrosia petiolata sporophytes in example 1.

TABLE 4 sporophyte transplant results

Example 2

To investigate the effect of different media on spore germination, the following steps were performed:

(1) selection and disinfection of pyrrosia pedunculata spores

Collecting mature sporophylls with full and robust sporangiums in Anshun city of Guizhou in 2018 for 10 months, slightly sweeping impurities on the surfaces of the sporophylls off by using a brush, collecting the sporophylls, filling the sporophylls into a sulfuric acid paper bag, and storing the sporophylls in a refrigerator at 4 ℃ for later use; then the sporophyll is placed inSterilizing on clean bench, soaking in 70% ethanol for 30s, washing in sterile water for 3 times, and adding 0.1 wt% HgCl₂Soaking in the solution for 8min, and washing with sterile water for 5 times; the surface water of the spore leaf is firstly absorbed by sterile filter paper, and then the spore powder is gently scraped off from the spore leaf by a sterile scalpel, so as to obtain the sterilized explant material.

(2) Pyrrosia peduncularis spore germination culture

Inoculating the sterilized explant material into germination culture media with different inorganic salt concentrations for pyrrosia pedunculata spore germination to obtain a flaky prothallium, wherein all the germination culture media are not added with sucrose, 6g of agar is added into each liter of germination culture media, and the pH value is 5.8. The culture temperature of germination is 25 ℃, the illumination intensity of a daylight lamp light source is 1000-. 5 flasks were inoculated with each medium and repeated 3 times, and spore germination was observed and recorded after 30 days. Germination time refers to the time when small green spots appear on the medium, and the germination results are shown in Table 5.

TABLE 5 Effect of different media on spore germination

As can be seen from the data in Table 5, the spores required the shortest germination time in 1/8 sucrose-free medium, the germination time was 15 days, and the germination rate was 100%; spores required the longest germination time in MS liquid medium, 28 days. The optimal germination medium of pyrrosia petiolata is 1/8MS medium + agar 6 g/L.

Example 3

To investigate the effect of different media and sucrose concentrations on the proliferation of prothallium, the following steps were performed:

transferring the sheet-shaped prothallium in the optimal germination culture medium (the optimal germination culture medium is 1/8MS culture medium + agar 6 g/L.) in the example 2 to a proliferation culture medium for proliferation to obtain a prothallium group; specifically is provided withGroup 8 contains sucrose solutions of different concentrations and B₅、1/2B₅6g of agar is added into each liter of culture medium of the proliferation culture medium combined by the culture medium, the pH value is 5.8, the culture temperature is 25 +/-2 ℃, the illumination intensity is 1000-1500lx under a daylight lamp light source, the illumination time is 12h/d, 5 bottles are inoculated in each treatment, the operation is repeated for 3 times, and the growth condition of the prothallium is observed and recorded after 30 days. Specific results are shown in table 6.

TABLE 6 Effect of different media and sucrose concentrations on Protozoa proliferation

As can be seen from the data in Table 6, 1/2B was added without sucrose₅、B₅In the culture medium, the growth situation of the prothallium is better, the color is bright green, but the growth speed is slow. Wherein, B₅+30g·L^-1The volume of the prothallium mass in the sucrose culture medium is larger, the growth speed is high, B₅+30g·L^-1The sucrose culture medium is the best culture medium for the proliferation of the prothallium.

Example 4

To investigate the effect of different light intensities on sporozoite induction, the following steps were performed:

placing vermiculite as culture medium in a high pressure steam sterilization pot at 121 deg.C for 30min, and oven drying the medium in an oven after sterilization. Before the seedling raising tray is used, a 5% sodium hypochlorite solution is used for surface disinfection, the sterilized matrix is filled in the seedling raising tray after the seedling raising tray is dried, the matrix is placed in the seedling raising tray and then compacted and leveled, and the whole tray is soaked in water until the surface of the matrix is wet. The proliferated prothallium mass (i.e., in B of example 3)₅+30g·L^-1A sucrose medium is a prothallium mass obtained by proliferating a prothallium) was taken out of a culture flask, the medium was washed with distilled water and transplanted into a substrate, the photoperiod was set at 12h/d, and the light intensity was set at 1000lx, 2000lx and 3000lx 3 for treatment.Spraying water once a day. Periodically observing the induction condition of the sporophyte, recording the initial time of the sporophyte formation and counting the induction rate. Sporulation time refers to the time from the mass of the original leaf until the appearance of the first true leaf. Specific results are shown in table 7.

TABLE 7 Effect of different light intensities on sporozoite Induction

As can be seen from Table 7, the sporozoite induction rate was the highest but the induction time was the longest when the light intensity reached 2000 lx. The optimum light intensity for induction of sporozoites was 2000 lx.

Example 5

To investigate the effect of different substrates on sporozoite transplantation, the following steps were performed:

the juvenile sporophyte mass obtained by induction (i.e., juvenile sporophyte mass obtained in example 4 in which the optimum illumination intensity of the induced sporophyte was 2000 lx) was separated into individual juvenile sporophytes, and the individual juvenile sporophytes were transplanted into the following 6 matrices: A. river sand; B. soil preparation; C. quartz sand; D. vermiculite; E. river sand: garden soil: pine needle soil is 1:1:3 (mass ratio); F. vermiculite: bark: perlite is 2:1:1 (mass ratio). Transplanting the juvenile sporophytes into the matrix at the row spacing of 2.5cm, watering the matrix thoroughly, covering the pot mouth with a plastic film, uncovering the film after the juvenile plants are adapted, and carrying out conventional management. After 30d, recording the survival rate of juvenile sporophytes. Specific results are shown in table 8.

TABLE 8 Effect of different substrates on sporozoite transplantation

As can be seen from table 8, in vermiculite: bark: in the perlite-2: 1:1 combined matrix, the survival rate after transplanting is as high as 100 percent; the survival rate of transplanting the garden soil matrix is the lowest, and is only 50%. Therefore, the most suitable substrate for sporophyte transplantation is vermiculite: bark: perlite is 2:1:1 composite matrix.

Example 6

The same procedure as in example 1 was repeated except that the matrix was pine needle soil in the case of transplanting into the matrix as compared with example 1.

Example 7

The same as example 1 except that the substrate was bark, which was transplanted into the substrate, was compared with example 1.

Example 8

The same as example 1, except that the substrate was perlite in the transplanting into the substrate, was compared with example 1.

Example 9

The procedure was as in example 1 except that the medium was river sand, garden soil, quartz sand, vermiculite, pine needle soil, bark, and perlite, and the medium was transplanted into the medium at a ratio of 1:1:1:1:1:1: 1.

Example 10

Example 11

The procedure of example 1 was repeated except that the substrate was changed to garden soil, quartz sand, vermiculite, pine needle soil, bark, and perlite in comparison with example 1, and the transplanting was performed into the substrate at a ratio of 1:1:1:1:1: 1.

Example 12

The procedure of example 1 was repeated except that the substrate was quartz sand, vermiculite, pine needle soil, bark, and perlite, which were transplanted into the substrate in example 1, and the procedure was otherwise the same as that of example 1.

Example 13

The procedure of example 1 was repeated except that the substrate was quartz sand, pine needle soil, bark, and perlite, 1:1:1, compared with example 1.

Example 14

The procedure of example 1 was repeated except that the substrate was quartz sand, pine needle soil and perlite in a ratio of 1:1:1, compared with example 1.

Example 15

The same procedure as in example 1 was repeated, except that the pH of the germination medium was 5.6 and that of the propagation medium was 5.6, as compared with example 1.

Example 16

The same procedure as in example 1 was repeated, except that the pH of the germination medium was 6.0 and that of the propagation medium was 6.0, as compared with example 1.

Example 17

The same procedure as in example 1 was repeated, except that the germination was carried out at a culture temperature of 27 ℃ under illumination with a fluorescent lamp light source at an illumination intensity of 1500lx for a period of 14h/d and the propagation was carried out at a culture temperature of 27 ℃ under illumination with a fluorescent lamp light source at an illumination intensity of 1500lx for a period of 14 h/d.

Example 18

The same procedure as in example 1 was repeated, except that the germination was carried out at 23 ℃ under the illumination of a fluorescent lamp light source at an illumination intensity of 1000lx for a period of 10h/d and the propagation was carried out at 23 ℃ under the illumination of a fluorescent lamp light source at an illumination intensity of 1000lx for a period of 10h/d, as compared with example 1.

Example 19

The procedure of example 1 was repeated, except that the light induction was carried out at 23 ℃ for 10 hours/day with a fluorescent lamp, the intensity of light was 1000lx, and water was sprayed twice a day in the light induction.

Example 20

The procedure of example 1 was repeated, except that the light induction was carried out at a culturing temperature of 27 ℃ by using a fluorescent lamp, the light time was 14h/d, the light intensity was 3000lx, and the water spray was carried out once a day in the light induction.

The beneficial effects of the invention are as follows, the embodiment of the invention provides a sporophyte propagation method with pyrrosia petiolata, which can shorten the induction period of sporophyte and improve the induction rate of sporophyte. The invention takes spores as explants, establishes a propagation system of pyrrosia peduncularis sporophytes, provides an effective method for protecting wild pyrrosia peduncularis resources, and simultaneously provides theoretical and technical support for industrial seedling culture of pyrrosia peduncularis; in the method for propagating the sporophyte of pyrrosia petiolata, a large amount of prothallium materials can be obtained by utilizing the characteristics of large quantity of pyrrosia petiolata spores and large propagation coefficient in the process of tissue culture, and an adequate material source is provided for transforming juvenile sporophytes. The sporophyte can be obtained through fertilization, however, sufficient water is not supplied to the sperm cells and the egg cells in the closed space, so that an incomplete tissue culture technology can be utilized, and a proper amount of water is provided for the fertilization process of the pyrrosia peduncularis sporophyte through a means of artificial water supplement, and the smooth implementation of the fertilization process is ensured. The propagation method of the pyrrosia peduncularis sporophyte can greatly shorten the formation period of the sporophyte by combining the tissue culture technology and the incomplete tissue culture technology, and can also improve the induction rate of the sporophyte, thereby obtaining a large number of pyrrosia peduncularis seedlings. The spore germination rate of the method reaches up to 100 percent, the spore induction rate reaches up to 85 percent, the survival rate of the transplanted sporophyte reaches 100 percent, and only 60 days are needed from the spore germination to the young sporophyte formation.

While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims

1. A sporophyte propagation method of pyrrosia petiolata, which is characterized by comprising the following steps:

1) selecting mature pyrrosia pedunculosa sporophyll, and obtaining spore powder from the pyrrosia pedunculosa sporophyll after disinfection to obtain a disinfected explant material;

2) inoculating the explant material into a germination culture medium for pyrrosia pedunculata spore germination to obtain a flaky prophyll; wherein the germination culture medium is a culture medium obtained by diluting an MS culture medium without adding sucrose;

3) transferring the sheet-shaped prothallium to a proliferation culture medium for proliferation to obtain a prothallium group; the proliferation medium is prepared by mixing sucrose solution with B₅Culture medium 1/2B₅Combining culture media to obtain;

4) and (3) carrying out illumination induction on the prothallium body group to obtain a juvenile sporophyte group.

2. The method for propagation of sporozoites of pyrrosia petiolata according to claim 1, wherein the pH of said germination medium is 5.6 to 6.0 and the pH of said propagation medium is 5.6 to 6.0.

3. The method for propagation of sporophytes of Potentilla anserina according to claim 1, wherein the germination is performed at a cultivation temperature of 25 ± 2 ℃ by using a daylight lamp light source with an illumination intensity of 1000-.

4. The method for propagation of sporophytes of Podostigma longissima as claimed in claim 1, wherein the concentration of said sucrose solution is 0-30 g-L^-1。

5. The method for propagation of sporophytes of Potentilla anserina according to claim 1, wherein the propagation is performed by illumination with a daylight lamp at a cultivation temperature of 25 ± 2 ℃ with an illumination intensity of 1000-.

6. The method for propagating sporophytes of pyrrosia petiolata according to claim 1, wherein the light induction is performed by using a daylight lamp light source at a culture temperature of 25 ± 2 ℃, the light time is 10-14h/d, the light intensity is 1000-.

7. A method of propagation of sporophytes of Podostigma longissima as claimed in claim 1, wherein said method of propagation of sporophytes of Podostigma longissima further comprises the step of exercising said prothallium mass before said photoinduction.

8. The method for propagation of sporophytes of Podostigma longissima as claimed in claim 1, further comprising the step of transplanting the juvenile sporophytes obtained by light induction into individual juvenile sporophytes, transplanting the individual juvenile sporophytes into a substrate, watering for conventional management to obtain Podostigma longissima plants.

9. A method of propagation of sporozoites of Podostigris as claimed in claim 8, wherein the substrate is any one or more of river sand, garden soil, quartz sand, vermiculite, pine needle soil, bark or perlite.

10. Use of a method of propagation of sporozoites of the species pyrrosia according to any of claims 1 to 9 in the cultivation of ferns.